Over 4 million individuals in the U.S. are infected with the hepatitis C virus (HCV). Essential mixed cryoglobulinemia (EMC) is an extra-hepatic manifestation of chronic HCV characterized by a systemic vasculitis resulting from production of cold-precipitable immunoglobulins largely composed of monoclonal (Type II) or polyclonal (Type III) IgM rheumatoid factors (RF). More than 80 percent of patients with EMC have chronic HCV and this complication of infection is difficult to treat and results in substantial morbidity. The pathogenesis of HCV-associated EMC is poorly understood and the antigenic stimulus for and site(s) of production of RF/cryoglobulins remain undefined. Hepatic lymphoid follicles (LF), present in approximately 90 percent of HCV patients are likely sites of generation of antibodies with RF and cryoglobulin activity. These LF resemble germinal centers, the sites of normal affinity maturation of the B cell response and contain B cells that are predominantly monoclonal, consistent with an antigen-driven response. In other chronic inflammatory conditions, similar LF in non-lymphoid tissue are known sites of antigen-driven B cell affinity maturation. Monoclonal RF (mRF)from patients with HCV-associated type II cryoglobulinemia have been found to bind HCV particles directly and infection with particular HCV genotypes appears to correlate with the presence and clonality of cryoglobulins, and clinical manifestations of EMC. These studies support the hypothesis that EMC results from mRF/cryoglobulin production in hepatic LF in response to chronic stimulation of a subset of B cells by HCV antigens. To test this hypothesis, the antigen specificity and clonality of individual B cells in particular hepatic LF from HCV patients with and without EMC must be assessed. We will utilize micromanipulation and single cell PCR techniques, to determine if a clonal/cryoglobulin B cell response occurs in hepatic LF, and whether particular HCV antigens drive antibody production and influence clinical manifestations. This proposal is directly applicable to RFA DK-98-017: Hepatitis C: Natural History, Pathogenesis, Therapy, and Prevention, as it addresses whether particular HCV antigens are involved in the pathogenesis of HCV-associated EMC.